There is much interest in the general field of the genetic engineering of living organisms. In the genetic engineering of an organism, foreign genetic material, typically a DNA vector constructed so as to express a suitable gene product in the cells of the target organism, is transferred into the genetic material of cells of the organism, through one of a variety of processes. In the past, the transformation techniques have varied widely from organism to organism, and few genetic transformation techniques have been developed which seem applicable to a large number of different organisms in different biological classes or kingdoms. Some of the prior art mechanisms utilized for inserting genetic material into living tissues include direct micro-injection; electroporation, a technique in which individual cells are subjected to an electric shock to cause those cells to take up DNA from a surrounding fluid; liposome-mediated transformations, in which DNA or other genetic material is encapsulated in bilipid vesicles which have an affinity to the cell walls of target organisms; and certain specific types of biological vectors or carriers which have the ability to transfect genetic material carried within them into certain specific target organisms, such as the plant transformation vector Agrobacterium tumefaciens and retroviral vectors which are used in animal hosts.
One technique exists which seems applicable to a wide range of target organisms. This technique is referred to as particle-mediated genetic transformation. In this technique, the genetic material, be it RNA or DNA, is coated onto small carrier particles. The carrier particles are then physically accelerated into the tissue which is to be transformed. For the process to work, the carrier particles are selected to be small enough so that they may be hurled through the walls and into the interior of cells of the target organism, without causing injury or significant harm to those cells. Several articles have been published describing the techniques and the apparatus utilized in such a particle-mediated transformation technique. Klein et al., "High-Velocity Microprojectiles for Delivering Nucleic Acids into Living Cells," Nature 327:70-73 (1987); and Sanford, "The Biolistic Process," TIBTECH, 6:299-302 (1988). Sanford and Klein, who are early investigators of particle-mediated transformation techniques, utilized a macro-projectile to accelerate the small carrier or micro-particles. The macro-projectile or macro-particle used by Sanford and Wolfe was literally a bullet fired by a ballistic shell which was, in actual fact, a firearm cartridge. The use of such extremely high velocity acceleration techniques required a large instrument, with very good shielding and a safety interlock, to prevent inadvertent harm to the experimenters.
A second technique developed for the acceleration of carrier particles carrying biological molecules into target cells for genetic transformations was based on a shock wave created by a high-voltage electric spark discharge. This apparatus, described in European published patent application number 270,356 and in U.S. Pat. No. 5,120,657, involves a pair of spaced electrodes placed on a spark discharge chamber. A high-voltage electric discharge is then passed between the electrodes to vaporize a water droplet placed between the electrodes. The spark discharge vaporizes the water droplet creating a shock wave, which accelerates a carrier sheet previously placed on the discharge chamber. The carrier sheet carries thereon the carrier particles, which have the biological genetic materials thereon. The carrier sheet is accelerated toward a retaining screen where the carrier sheet is stopped, the particles are separated from it, and only the carrier particles pass on into the biological tissues. The design for the particle acceleration apparatus as described in these publications was one which involved the desk top, or bench top, apparatus of relatively significant size and complexity and which was relatively immobile.
A smaller particle acceleration apparatus in which the operative portion of the device is hand-held is described in U.S. Pat. No. 5,149,655. The hand-held device permits the acceleration of particles carrying biological molecules into whole living organisms that are larger than can readily be placed onto a bench top unit.
The various particle acceleration techniques and devices described above were developed primarily for genetic transformation of individual cells and plant tissue. However, as the particle acceleration technology has developed, its use with laboratory and domestic animals has become an increasingly important aspect of the technology. Moreover, particle acceleration is proving to be well suited to developing genetic therapies and genetic vaccines in large animals and humans.
The underlying goal of particle acceleration is the efficient transfer, uptake and expression of foreign genetic material in a target animal, plant, cell, or tissue. Typically, the genetic material transferred is DNA encoding a peptide or protein absent from the target cells. The transferred DNA typically includes the necessary transcription and translation regulatory elements such as promoters, terminators, and ribosome binding sites, as required by a particular target host.
In plants, plant cells, and some animals much effort has been directed to obtaining germ-line transformants capable of constitutively or inducibly expressing a desirable gene product, and to transferring that capability to progeny plants grown from seed.
In contrast, the focus of more recent developments in human genetic therapy and vaccination has been the transient expression of transferred genes, either to provide a therapeutic protein to a somatic tissue, or to induce an immune response to the product of the transferred gene. This latter approach is now believed to offer dramatic advantages over conventional immunization protocols, for several reasons.
First, it will be dramatically less expensive to produce stable vaccines made of DNA than to produce peptide- or protein-based vaccines. Second, the ability to easily modify DNA will likely permit the production of customized vaccines. Third, as more and more genes encoding medically important antigens are discovered, it will be possible to generate vaccines against a vast array of infectious agents for which no vaccines are presently available. Finally, recent observations indicate that particle acceleration of DNA into human skin or mucosal tissue is particularly well suited to inducing a long-lasting immune response. As such, rapid, needle-free, painless, non-invasive vaccination will be facilitated by bringing the principles of particle acceleration to bear upon the epidemiological problems of vaccinating large populations against an array of infectious agents.
As a result of the direction in which the particle acceleration technology has developed, the ability to deliver samples in rapid succession is now required. Whereas the time between samples was relatively unimportant when the target was a plant cell suspension, when treating a number of human beings or large animals, it is desirable to complete the particle acceleration protocol as quickly as possible. Particularly with large animals, it is desirable to restrain the target animals for as short a time as possible. In existing particle acceleration devices, it has been necessary to disassemble at least a portion of the device between each use to install a fresh sample carrier sheet and to clean the retaining screen. This would result in an unacceptably long delay between samples.
In addition, the carrier sheet was attached directly to the discharge opening of earlier particle acceleration devices, typically by relying on the surface tension of a liquid such as oil or water. An additional source of delay between samples arose from the need to provide new liquid to attach each corner of the sheet.
It is, therefore, desirable to provide a safe and effective particle acceleration device which allows for reliable, rapid, repetitive delivery of genetic material into targets with minimal time between samples.